Quantitative determination of gene expression levels is a powerful approach for the comparative analysis of normal and neoplastic tissue. Gene expression profiling is increasingly important both in biological research and in clinical practice and has been used to, for example, classify various cancer types, and to predict clinical outcome of cancer, such as breast cancer and lung cancer.
Analysis of gene expression at the mRNA level is a central component of molecular profiling. Sensitive and specific methods for studying RNA derived from fresh tissues and cells are well described, and include techniques based on the use of reverse transcriptase-polymerase chain reaction (RT-PCR). Recent technological improvements, including the introduction of highly sensitive fluorescence-based real-time RT-PCR procedures, now allow for rapid and specific quantification of even small amounts of mRNA. However, the use of RT-PCR based methods to quantify mRNA in clinical specimens has been restricted by the limited availability of suitable fresh or frozen study tissues. In many situations where gene expression profiling is potentially useful, there is insufficient material for analysis. To allow conclusions regarding the clinical significance of the results obtained with such techniques, the examination of large numbers of pathological tissue specimens representing different disease stages and histological tumour types and grades is essential.
One possible answer to this problem may lie in the archives of formalin-fixed, paraffin-embedded (FFPE) tissue specimens which have been archived in quantity in pathology departments, along with their clinical histories and prognoses throughout the world. These collections already represent an invaluable research resource for studying the molecular basis of disease, making it possible to perform large retrospective studies correlating molecular features with therapeutic response and clinical outcome. Accordingly, formalin-fixed samples are attracting increasing attention as RNA sources. Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, in conjunction with clinical data are the most widely available basis for such retrospective studies. The reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissue, however, has been subject to serious limitations so far.
Techniques for extraction and analysis of DNA from FFPE tissues have been optimized allowing a range of molecular genetic studies to be performed on archival and routine diagnostic histopathological material. Although this is a lesser problem for DNA, RNA isolated from paraffin-embedded tissue blocks is of poor quality because extensive degradation of RNA can occur before completion of the formalin fixation process. Moreover, formalin fixation causes cross-linkage between nucleic acids and proteins and covalently modifies RNA by the addition of mono-methylol groups to the bases. As a result, the molecules are rigid and susceptible to mechanical shearing, and the cross-links may compromise subsequent RNA extraction, reverse transcription and quantification analysis. Therefore, in order to utilize FFPE tissues as a source for gene expression analysis, a reliable method is required for extraction of RNA from the cross-linked matrix.
Since Rupp (Rupp, G. M. and Locker, J., 1988) first reported northern hybridization of formalin-fixed samples, significant efforts have been made toward recovery of RNA from formalin-fixed tissues. Various modifications were made to the extraction steps, using RT-PCR to evaluate the outcome. In all reports, successful amplifications were limited to small fragments and sensitivities in transcript detection were much worse than with fresh material, although their alterations to the protocols did improve the results somewhat. The following three possibilities have been stated as the reasons for the poor results: RNA was degraded in the tissue before, during or after fixation; the RNA was resistant to extraction probably due to cross-linking with proteins; the extracted RNA from fixed specimens was chemically modified by formalin in a way that is still elusive. However, direct evidence for each of these possibilities and thoughtful investigation regarding the contribution of these three possibilities to the overall results has been lacking.
U.S. Pat. No. 6,248,535 discloses a method for the isolation of RNA from formalin-fixed, paraffin-embedded (FFPE) tissue specimens. In such method, the tissue sample is first deparaffinized and further homogenized in a solution comprising a chaotropic agent, like, for example, guanidinium isothiocyanate. The homogenate is thereafter heated at about 100° C. in a solution with a chaotropic agent. RNA is further recovered from the solution by, for example, phenol-chloroform extraction.
Krafft et al. (1997) Molecular Diagnosis vol. 2, no. 3, pages 217-230 describes the isolation and amplification of RNA from FFPE tissue. A process is described comprising a digestion with proteinase K, followed by alcohol precipitation and RT-PCR. Several concentrations of proteinase K were tested; optimal proteolytic digestion was obtained with high concentrations of proteinase K.
Masuda et al. (1999) Nucleic Acid Research vol. 27, no. 22, pages 4436-4443 describe several methods for the extraction of RNA from formalin fixed samples, finding that a proteinase K digestion at 45° C. for one hour, followed by precipitation with alcohol and a treatment with DNAse.
Spetch et al. (2001) American Journal of Pathology vol. 158, no. 2, pages 419-429 describes a procedure for the quantitative gene expression analysis in microdissected archived formalin-fixed and paraffin embedded tumour tissue trough RNA micro-scale extraction in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction.
Finke, J. et al (1993). Biotechniques, Informa Life Sciences, vol. 14, no. 3, pages 448-453 describes a strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.
However, the above methods do not provide enough sensibility when small amounts of RNA are to be detected. More accurate and reliable techniques for the isolation of RNA from paraffin-embedded tissue are particularly needed for the study of gene expression in tumour tissues. The ability to routinely study mRNA expression in FFPE tumour tissues, even when only small amounts are present, would be an important advance, opening up the histopathology archive to molecular profiling and allowing analysis of gene expression at the RNA level in standard diagnostic specimens and allowing establishing good correlations between gene expression and the clinical outcome.